Reaction mass of 2-hexadecyl-N-(2-hydroxyethyl)-3-oxoicosanamide, 2-hexadecyl-N-(2-hydroxyethyl)-3-oxo-, N-(2-hydroxyethyl)-3-oxo-2-tetradecylicosanamide and N-(2-hydroxyethyl)-3-oxo-2-tetradecyloctadecanamide.

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 16 August 2011 and 15 September 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

PC-9S

IUPAC Name:

PC-9S

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor's identification: PC-9S
Description: off white powder
Purity: C16 - C16 = 10.2%, C16 - C18 C18 - C16 = 39.6%, C18 - C18 = 50.2%
Batch number: PS101103
Date recieved: 11 July 2011
Expiry date: 18 November 2012
Storage conditions: room temperature in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 15 August 2011 from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of inoculum:
The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.4 g/I prior to use.

Duration of test (contact time):

28 d

Details on study design:

TEST CONDITIONS
- Culture medium: Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 ml of culture medium and 26.5 ml of inoculum and aerated overnight. On Day 0 the test and reference items were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
- Test temperature: 21 deg C
- pH: 7.4
- Aeration of dilution water: yes overnight, The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
- Continuous darkness: yes
- Culture medium: The following 4 solutions was added to 1L of purified water
10 ml of Solution a:
Solution A was made up of:
KH2P04 - 8.50g/l
K2HP04 - 21.75 g/I
Na2HP04.2H20 - 33.40 g/I
NH4CI - 0.50 g/I
pH = 7.4

1 ml of Solution b: CaCI2 - 27.50 g/l
1 ml of Solution c: MgS04.7H20 - 22.50 g/l
1ml of Solution d: FeCI3.6H20 - 0.25 g/l

TEST SYSTEM
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The reference item (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
c) The test item, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
d) The test item plus the reference item in inoculated culture medium to give a final concentration of 20 mg carbon/l to act as a toxicity control (one vessel only).

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/1. The test was carried out in a temperature controlled room at approximately 21°C, in darkness.

The CO2 produced by degradation was collected in two 500 ml Dreschel bottles
containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using
purified de-gassed water.

SAMPLING
Samples (2 ml) were taken from the control, reference and test item first CO2 absorber vessels on Days 0, 3, 6, 8, 10, 14, 21, 28 and 29 and from the toxicity control first CO2 absorber vessel on Days 0, 3, 6, 8, 10 and 14. The se~ond absorber vessel was sampled on Days 0 and 29 for the control, reference and test item and on Day 0 for the toxicity control.

The samples taken on Days 0, 3, 6, 8, 10, 14, 21, 28 and 29 were analysed for CO2 immediately.

On Day 28, 1 ml of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.

CONTROL AND BLANK SYSTEM
- Toxicity control: For the purposes of the test, a toxicity control, containing .the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.

An amount of test item (39.3 mg) was dispersed in approximately 400 ml of culture medium with the aid of high shear mixing (approximately 7500 rpm for 15 minutes) prior to dispersal in inoculated culture medium. An aliquot (51.4 ml) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 litres to give a final concentration of 13.1 mg test item/I plus 17.1 mg sodium benzoate/I, equivalent to a total of 20 mg carbon/I.

Statistical analysis:
Statistical analysis of the Day 29 IC values for the control and test item vessels was carried out using a Student's t-test to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Details on results:

Inorganic carbon values for the test item, reference item, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3, and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 4. The pH values of the test preparations on Day 28 are given in Table 5. Observations made on the contents of the test vessels are given in Table 6.

Tables 1 to 6 can be found in the attached background material section and figure 1 can be found in the illustration (picture/graph attachments section)

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 70% degradation after 14 days and 77% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

The total CO2 evolution in the control vessels on Day 28 was 28.73 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

The difference between the values for CO2 production at the end of the test for the replicate vessels was < 20% and satisfied the validation criteria given in the OECD guidelines.

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of control replicate R2, reference replicate R2 and test item replicate R1.

Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were no statistically significant differences (P≥ 0.05) between the control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.

The toxicity control attained 40% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Please see info on any other information on results section for more details

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 B.

Executive summary:

Introduction: A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301 B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M)).

Methods. The test item, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days. The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results. The test item attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 B.